Vol. 34, No. 6, June 1969
ACTIONOF HYDROGEN FLUORIDE ON PHOSPHATE ESTERS 1539
After drying (MgS04), the chloroform solution waa concentrated and the residue distilled to yield the products. Diethyl cyacetoxy-8-acetamidoethylphosphonate (19) [bp 160” (0.25 mm) ; ir (film) 3300, 3080 (NH), 1745 (ester C=O), 1660 (amide C=O), 1220 (P===O), 1158 (POCzH5), 1018 cm-’ (PO-alkyl)] was obtained from 17 (30% yield from 3a) and diethyl 8-acetoxyr-acetamidopropylphosphonate (25) [bp 162” (0.25 mm) ; ir (film) 3300,3075 (NH), 1735 (ester C=O), 1682 (amide C=O), 1217 (P=O), 1170 (POCzH,), 1020 cm-l (PO-alkyl)] was obtained from 24 (75% yield from 4 ) .
Registry No.-3b, 19462-37-4; 19462-39-6; 13, 19462-40-9; 19462-42-1; 16, 19462-43-2; 19462-45-4; 20, 1866-28-0; 19462-48-7; 23, 19462-49-8;
7a, 19462-38-5; 14, 19462-41-0; 18, 19462-44-3; 21, 19462-47-6; 24, 19462-50-1;
7b, 15, 19,
22, 25,
19462-51-2; 26, 19462-52-3; 27, 19462-53-4; 19462-54-5; 29, 19462-45-4; 30, 19462-56-7.
28,
Acknowledgment.-Partial support for these studies was provided by grants from the Gulf Oil Foundation and NASA (to the University of Pittsburgh). Pmr studies were performed using, in part, the NMR Facility for Biomedical Studies supported by a grant (FR-00292-03) from the National Institutes of Health. We are indebted to Mr. R. H. Chodosch for preliminary experiments, Messrs. T. Finkelstein and E. P. Mazzola for assistance in the determination of spectra, Dr. J. A. Ross for providing information in advance of publication, and Dr. E. D. Weil for helpful suggestions.
The Action of Hydrogen Fluoride on Nucleotides and Other Esters of Phosphorus(V) Acidslv2 DAVIDLIPKIN,BRUCEE. PHILLIPS, AND JOHN W. ABRELL Department of Chemistry, Washington University, St. Louis, Missouri 68150 Received October 9, 1968
The action of hydrogen fluoride, both liquid and aqueous, on a number of mono- and diesters of orthophosphoric acid, two cyclic phosphates, mono and diesters of polyphosphates, phosphorofluoridate esters, and inorganic phosphates was investigated. The particular reactions which take place are found to be a function of temperature (-50 to +25”), time (0.04-28 hr), and acid concentration. A comparison of the acid-catalyzed reactions of phosphate esters in aqueous solution with the behavior of such compounds toward 607, hydrofluoric acid brings out several interesting contrasts. First, the reactions in 60% hydrofluoric acid which are described here are fast compared with reactions observed with ordinary aqueous acids. Second, in the reactions with 60% hydrofluoric acid all of the evidence points toward the conclusion that phosphorus-oxygen, rather than carbon-oxygen, bond cleavage takes place exclusively. Third, this acid is a highly specific dephosphorylating agent compared with ordinary aqueous acids. These three features of the chemical properties of hydrogen fluoride in relation to phosphorus(V) esters are correlated with information concerning the characteristics of hydrogen fluoride both aa an anhydrous liquid and in concentrated aqueous solution. Since extended exposure to 60% hydrofluoric acid causes no deamination of adenine, guanine, or cytosine, a method was devised for the base analysis of ribonucleic acid (RNA) by degradation with this reagent.
Hydrogen fluoride, either as anhydrous liquid or aqueous solution (hydrofluoric acid), has been used relatively little in nucleotide chemistry in comparison with other areas of organic Chemistry? The structure proofs of A-3’: 5’-PdJ and of the diastereoisomeric (1) (a) This investigation was supported, in part, by Public Health Service Research Grant No. CA-03870 from the National Cancer Institute and, in part, by Grant No. GB-2980 from the National Science Foundation. (b) The major portion of this paper is based on the Ph.D. dissertation of J. W. Abrell, Washington University, St. Louis, Mo., 1965. (2) For a brief summary of the results of this investigation, see D. Lipkin, J. W. Abrell, and B. E. Phillips, Abstracts, Seventh International Congress of Biochemistry, Tokyo, Aug 1967, No. B-57, p 631. (3) K. Wiechert in “Newer Methods of Preparative Organic Chemistry,” Interscience Publishers, New York, N. Y., 1948, pp 315-368. (4) The abbreviations used follow: A-2’ (3’)-P, adenosine 2’(3‘)-phosphate; A-5’-P, adenosine 5’-phosphate; A-2’:3‘-P, adenosine 2‘:3’- (cyclic) phosphate; A3’:5‘-P, adenosine 3’:5’- (cyclic) phosphate; A-5’-PF, adenosine 5’-phosphorofluoridate; ADP, adenosine 5’-diphosphate: APPA, P1,Pkliadenosine 5’;pyrophosphate; ATP, adenosine 5’-triphosphate: C-2’(3’)-P, cytidine 2’(3’)-phosphate; dA-5‘-P, 2’deoxyadenosine 5’-phosphate; DCC, dicyclohexylcarbodiimide; DNA, deoxyribonucleic acid; DPN, diphosphopyridine nucleotide; 2-dR-5-P, 2deoxyribose 5-phosphate: G-2’(3’)-P. guanosine 2’(3‘)-phosphate; Me A-5’-P, methyl ester of adenosine 5’-phosphate; Nam, nicotinamide; NR, nicotinamide riboside; NR-5‘-P. nicotinamide riboside 5’-phosphate; NR-5’-PF. nicotinamide riboside 5’-phosphorofluoridate; RNA, ribonucleic acid: T3‘-P, thymidine 3’-phosphate; T-5’-P, thymidine 5’phosphate; Tris, tris(hydroxymethyl)aminomethane; U-2’(3’)-P, uridine 2’(3’)-phosphate. (5) (a) D. Lipkin, R. Markham, and W. H. Cook, J . A m . Cham. SOC., 81. 6075 (1959): (b) D. Lipkin. W. H. Cook, and R. Markham, ;bid., Si, 6198 (1959).
2’: 3’-benzylidene ribonucleosides6 were dependent , in part, on degradations carried out with liquid hydrogen fluoride and hydrofluoric acid, respectively. Furthermore, 5-iodouridine 5‘-triphosphate has been degraded with 60% hydrofluoric acid to 5-iodouridine 5’-phosphate and then to 5-iodouridine.’ The principal objective of this study was to investigate, in detail, the a.ction of hydrogen fluoride on ordinary mononucleotides. The study includes, however, a broader spectrum of phosphorus compounds. It covers a number of mono- and diesters of orthophosphoric acid, two cyclic phosphates, mono- and diesters of polyphosphates, phosphorofluoridate esters, and inorganic phosphates. That the present results are of rather general interest is attested to by the fact that they already have been utilized to obtain structural information concerning the teichoic acids.8 Results and Discussion Various acid-catalyzed reactions have been observed for nucleoside monophosphates in aqueous solution. (6) (a) D. Lipkin, B. Phillips, and W. H. Hunter, Tetrahedron Lett., No. 21, 18 (1959); (b) B. E. Phillips, Ph.D. Thesis, Washington University, St. Louis, Mo.,1961. (7) D. Lipkin, F. B. Howard, D. Nowotny, and M. Sano, J. B i d . Chem., 988, PC2249 (1963). (8) L. Glaser and M. M. Burger, ibid., 239, 3187 (1964).
1540 LIPKIN, PHILLIPS, AND
-Substrate
ABRELL
The Journal of Organic Chemistry
TABLEI DEGRADATION OF NUCLEOSIDE MONOPHOSPHATES' Reaction conditionsTime, hr Temp, O C
Separation methodb
Nucleotide, %
Reaction products Nucleoside, %
Base, %
Loss. %
A-3'-Po A-2'(3')-P
I -23 D 42 58 0.5 0 A 4 84 10 2 28" 25 A 94 6 A-5'-P 1 - 23 A 53 43 4 0.5 0 A 13 73 7 7 14" 25 A 102, A-5'-PF 0.25 0 B 700 30 Trace Me A-5'-P 1 0 A 63h 26 4 7 C-2'(3')-P 1 0 A 96 4 17' 25 Borate 86 14 G-2'(3')-P 0.5 0 Wateri Trace 76 13
